KR101501033B1 - Streptomyces koyangensis BIG11003 having nematicidal effect and uses thereof - Google Patents

Abstract

The present invention relates to Streptomyces koyangensis BIG11003 (KACC 91843P) with protease producing ability isolated from the soil. A protease produced by the Streptomyces koyangensis BIG11003 according to the present invention suppresses the generation of egg sacs on roots of the root-knot nematode and facilitates the growth of plants as well as suppresses the hatching of root-knot nematode and reduces mobility of larvae, and thus can be provided as a microorganism agent for an effective nematicidal action, which can substitute for chemical synthetic pesticides.

Description

Streptomyces strain BIG11003 strain having a nematicidal effect and its use {Streptomyces koyangensis BIG11003 having nematicidal effect and uses thereof}

The present invention relates to a Streptomyces Goyansis strain BIG11003 having a fungicidal effect and a use thereof, and more particularly to a strain Streptomyces koyangensis strain BIG11003 having a protease- (KACC 91843P); A composition for preventing or treating infection of a plant pathogenic nematocyst comprising the strain and an agrochemically acceptable carrier; A method of infecting or treating a plant pathogenic nematum by treating the composition with a plant part, soil or seed; And a phytopathogenic nematode controlling agent containing the composition as an active ingredient.

Damage caused by root nodule nematodes in soil has been a frequent cause of damage in domestic facilities. Especially, fruit and vegetable crops such as watermelon, melon, cucumber, etc. Damage caused by root-knot nematodes appears seriously in the cultivation area, and there is a problem that it is difficult to use the control methods such as fresh water and rotation in such areas. Control of nematodes by chemical methods in the control of root-knot nematodes limits its use globally due to the danger of residual pesticides and ecosystem destruction since the drugs are toxic or very toxic. In addition, many studies have been carried out to prevent damage to root-knot nematodes, but most studies are concentrated on chemical insecticides and plant extracts.

Among the methods of controlling nematodes, soil disinfection using solar heat in the plantation area is not widely used due to the seasonal limitation that can be carried out only in July and August, the prolongation of disinfection period and the low mortality effect at the time of planting.

As the demand for domestic organic and environmentally friendly agricultural products is increasing, interest of microbial pesticides is growing as the only means for farmers to replace chemical synthetic pesticides. However, microbial preparations for soil pollution control, which are produced as eco-friendly organic materials in Korea, Compared to the absolute lack.

Korean Patent No. 0860726 discloses 'Bacillus Surinjiniensis Probio-35 and an effective method for controlling nematodes using the same,' Korean Patent No. 0791983 discloses a microorganism belonging to the genus Bacillus subtilis, A microorganism preparation for controlling nematodes for producing nematodes ", but the Streptomyces Goysungi strain BIG11003 strain having the nematode effect of the present invention and its use have not been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method for producing a proteolytic enzyme from Streptomyces The present inventors have completed the present invention by confirming that the strain BIG11003 was selected and identified, and that when the above-mentioned strain and a plant extract having excellent nematocidal effect were mixed, it had an effect of preventing and treating root-knot nematodes.

In order to solve the above problems, the present invention provides Streptomyces koyangensis BIG11003 strain (KACC 91843P) having a protease-producing ability separated from soil.

The present invention also provides a composition for preventing or treating infection of a plant pathogenic nematocyst comprising the strain and an agrochemically acceptable carrier.

The present invention also provides a method for infecting or treating a plant pathogenic nematism by treating the composition with a plant part, soil or seed.

The present invention also provides a plant pathogenic nematocide containing the composition as an active ingredient.

Streptomyces Enhancement N-Sys according to the invention (Streptomyces The protease produced by the BIG11003 strain inhibits egg hatching of the root-knot nematode and reduces the motility of the larva, as well as inhibits the formation of the oviduct in the root by the root-knot nematode and smoothes the growth of the plant , Can be provided as an effective microorganism preparation for controlling nematode that can replace chemical synthetic pesticides.

1 is a Streptomyces Enhancement N-Sys (Streptomyces having the proteolytic enzyme (protease) producing ability of the present invention isolated from soil lt ; RTI ID = 0.0 > (KACC 91843P). < / RTI >
Fig. 2 is a diagram showing the phylogenetic tree constructed through comparison of 16S rDNA nucleotide sequence showing the relationship between BIG11003 strain and other related strains.
FIG. 3 is a graph showing the results of pretreatment of a control agent containing the BIG11003 strain of the present invention as an active ingredient and confirming root development after 10 weeks in tomatoes inoculated with root-knot nematode larvae in order to confirm the pre- to be. DW: Fosthiazate as a negative control, Fosthiazate: Experimental group treated with 250 ppm of fosthiazate used as a nematode control, Liquid formulation (1000 ppm): 1000 ppm treatment of the pesticide of the present invention, (2000ppm): Treatment of 2,000 ppm of the pesticide of the present invention.
FIG. 4 shows the result of pretreatment of a control agent containing the BIG11003 strain of the present invention as an active ingredient to confirm the effect of pre-treatment on the nematode, to be. DW: Fosthiazate as a negative control, Fosthiazate: Experimental group treated with 250 ppm of fosthiazate used as a nematode control, Liquid formulation (1000 ppm): 1000 ppm treatment of the pesticide of the present invention, (2000ppm): Treatment of 2,000 ppm of the pesticide of the present invention.
FIG. 5 is a graph showing the effect of controlling the bacterium production inhibition on the tomato nematode control according to the present invention. It is a picture. DW: Fosthiazate as a negative control, Fosthiazate: Experimental group treated with 250 ppm of fosthiazate as a nematode used as a positive control, Liquid formulation (1000 ppm): 1000 ppm of a pesticide of the present invention.
FIG. 6 is a graph showing the effect of the control agent containing the BIG11003 strain of the present invention as an active ingredient on tomatoes formed by root-knot nematodes and capillaries by root-knot nematode, to be. DW: Fosthiazate as a negative control, Fosthiazate: Experimental group treated with 250 ppm of fosthiazate as a nematode used as a positive control, Liquid formulation (1000 ppm): 1000 ppm of a pesticide of the present invention.

In order to accomplish the object of the present invention, the present invention provides Streptomyces koyangensis BIG11003 strain (KACC 91843P) having a protease-producing ability separated from soil.

The strain Streptomyces koyangensis strain BIG11003 (KACC 91843P) was isolated from the soil of a cucumber cultivar. In addition to the effect of inhibiting egg hatching of the root-knot nematode and reducing the motility of the larvae, the strain Streptomyces koyangensis strain BIG11003 It inhibits the production of oocysts and promotes the growth of plants. The strain Streptomyces koyangensis strain BIG11003 was deposited at the Korean Agricultural Culture Collection (KACC) on Jul. 11, 2013 (Accession No .: KACC 91843P).

Streptomyces stimulus strain BIG11003 strain according to an embodiment of the present invention may have carnivorous activity.

According to one embodiment of the present invention, the nematode activity of Streptomyces stimulant strain BIG11003 is generally caused by a pathogenic nematode ( Meloidogyne spp.); Sister nematodes such as Heterodera spp., Globodera spp. And the like; Primateen (Pratylenchus.); Tylenchulus semipenetrans ; Radopholus; Ditylenchus; Anguina; It is a live nematode activity against Aphelenchoides, preferably a live nematode activity against the root nematode ( Meloidogyne sp.), But is not limited thereto.

The present invention also provides a composition for preventing or treating infection with a phytopathogenic nematode comprising Streptomyces koyangensis strain BIG11003 of the present invention and a pesticide acceptable carrier.

The composition for preventing or treating infectious disease of phytopathogenic nematodes according to the present invention may be prepared according to a method of microbial pesticide formulation in a manner known in the art, for example, a culture of the cultured microbial cells, lyophilized microbial cells, starch, crude protein, , Pellets or granules, microcapsules, etc., and they can be used as they are. The composition can be obtained by adding other components which are easy to apply according to the target nematodes and crops such as surface activators, inert carriers, preservatives, humectants, feed accelerators, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectants, can do. The compositions of the present invention may be in a form or concentrate or a primary composition suitable for direct application by dilution with an appropriate amount of water or other diluent prior to application. It will be clearly understood by those skilled in the art that the pesticidal concentration may vary depending on the nature of the particular formulation, the type of application plant, the soil and the climate in the cultivation area, and the like, particularly concentrated or direct use.

The composition for preventing or treating infectious diseases of phytopathogenic nematodes according to the present invention is particularly useful for the prevention of the infection of plant necrotic nematodes because it grows at high density in the soil such as facility cultivation area where the sequence is performed and invades the roots of crops to slow the growth of crops and reduce the harvest, Since it can be used in the control of phytopathogenic soil nematodes, it is of particular interest that pollution, safety agricultural products are increasingly in demand, and the residual toxicity due to the use of pesticides and the awareness of environmental pollution are increasing. It can contribute greatly to the production of agricultural products.

The present invention also relates to a method for preventing or treating infectious diseases of phytopathogenic nematodes comprising Streptomyces koyangensis strain BIG11003 of the present invention and a pesticidally acceptable carrier by treating plant parts, A method for preventing or treating an infection of a pathogenic nematode is provided.

The composition for preventing or treating infectious disease of phytopathogenic nematodes according to the present invention is a composition for preventing or treating nematode infection of crops such as wheat, barley, rye, oats, rice Such as spinach, lettuce, asparagus, cabbage and brassica, carrots, onions, carrots and related crops), sugar beets (e.g., sugar beets and feed beets), cucumber plants Tomatoes, potatoes), citrus fruits (e.g., orange, lemon, grapefruit, Chinese mandarin), fruits such as apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries, (Eg, cotton, mustard seed, poppy, olive, sunflower seed, coconut, castor oil plant, cocoa seed, peanut), alfalfa, Probably, hemp, jute), (For example, corn, grass plants, tobacco, etc.), deciduous trees (for example, corn, chestnut, (For example, but not limited to, nuts, coffee, sugarcane, tea, vine, hops, bananas and natural rubber trees) or to the cultivated soil or seeds of these plants. Or less. On the other hand, in the case where it is necessary to prevent or treat nematode infestation in plant parts, soil, seeds, or where soil purification by removal of nematode is necessary, microorganisms having nematicidal activity of the present invention and / It may be possible to suppress the damage caused by nematodes by administering both microorganisms and chemical insecticides simultaneously.

The present invention also relates to a phytopathogenic nematode controlling agent comprising, as an active ingredient, a composition for preventing or treating infection of a plant pathogenic nematode comprising Streptomyces koyangensis strain BIG11003 of the present invention and a pesticide acceptable carrier Lt; / RTI >

The plant pathogenic nematode controlling agent according to one embodiment of the present invention may further include, but is not limited to, one or more of Azadirachta indica extract and Sophora angustifolia extract.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. Protease Productive  Branch BIG11003  Strain isolation and identification

In order to obtain a new strain exhibiting a controlling effect on root-knot nematodes, BIG11003 strain of Streptomyces sp. Having high protease activity from domestic cucumber cultivated soil was isolated pure (Fig. 1). In order to identify the strain isolated pure, the DNA of strain BIG11003 was extracted and the PCR amplified product for 16S rDNA was purified and used for analysis. The analyzed sequence (1,342 bp) was subjected to homology search in the database of DDBJ / NCBI / Gnenbank and RDP-II (ribosomal database project II). As a result, the strain containing the species of Streptomyces sp. as a strain belonging to the chemical group exhibits a flexible relationship Streptomyces Enhancement N-Sys (Streptomyces koyangensis) VK-60T ( AY079156) and 99.9%, BIG11003 strain having the protease producing ability of the present invention is Streptomyces Enhancement N-Sys (Streptomyces kangangensis ) species.

Example  2. The present invention BIG11003  Of strains and plant extracts Root-knot nematode  Reduction of larval mobility and inhibition of egg hatching

Streptomyces koyangensis strain BIG11003 isolated from Example 1 showed low proteolytic enzyme activity when cultured in PD or nutrient medium. However, when cultivated in bran medium, proteolytic enzyme producing root-knot nematode And an enzyme activity assay of Anson was modified to determine the protease activity. As a result, it was confirmed that the enzyme activity was 610 unit / ㎖. Or less, Streptomyces Enhancement N-Sys (Streptomyces The strain BIG11003 refers to a culture medium of Streptomyces koyangensis BIG11003 strain cultivated in a bran medium and contains a proteolytic enzyme secreted by the Streptomyces stimulant strain BIG11003.

Insecticides and fungicides for the development was performed an analysis of 190 species of plant extracts, as target material for more than 20 kinds of plant extracts secured confirming the mobility reduction of root-knot nematode larvae result, s (Azadirachta indica ) and Sophora angustifolia seed extracts were found to be effective against root nematode.

Thus, the inventive Streptomyces koyangensis) BIG11003 strain's extracts (Azadirachta indica ) and Sophora angustifolia (Rootgrass nematode larvae).

Nem extract ( Azadirachta indica ) was obtained by pulverizing 100 kg of Neem bark into powder and treating it with 700 kg of 100% ethanol and 300 kg of water for 6 hours. The concentration of the extracted nimble extract is 5,000 ppm. The seed extract of Phellinus linteus was obtained by pulverizing 6,100 kg of nursery seeds and shaking it in 3,000 kg of 100% ethanol and 1,000 kg of water. The concentration of extracted Pusan seed extract is 6,000 ppm.

Roots and infected soil of root - knot nematode plants were collected from Daejeon cucumber growing area, Gangwon - do tomato growing area and Chungnam melon growing area where the damage of root - knot nematode was serious and used for the experiment. In order to isolate nematodes from nematode infested with root nematode, soil samples collected were mixed well, and then impurities were extracted using a 50 mesh sieve. The sieved soil was poured into a deformation funnel having a diameter of 15 cm And treated at room temperature for 48 hours to isolate the root nematode larvae. The nematode solution, which had been separated from the transformant funnel, was filtered through a 500 mesh sieve and collected in a 15 ml tube. The nematocysts were allowed to stand at room temperature for 1 hour, and then 100 ng / ml or more of nematodes were inoculated into a 24-well plate. Streptomyces koyangensis ) BIG11003 strain, ( Azadirachta indica ) Extracts and ginseng ( Sophora angustifolia ) seed extracts were treated and evaluated after 4 days by changes in motility and morphology of nematodes. Effectiveness test was performed with three replicates. Fosthiazate, a registered root - knot nematode control agent, was used as a control. As a result, it was confirmed that the motility reduction effect was exhibited in all of the above treatments. In particular, BIG11003 strain and Pseudomonas aeruginosa seed extract were found to be more effective in decreasing motility of larva than that of the pesticide zoster (Table 1).

Reduction of motility of nematode larvae Processing contents Treatment concentration (ppm) Reduction of larval mobility (%) Ⅰ repeat Ⅱ repeat Ⅲ repeat Average S. koyangensis BIG11003 strain 2,000 40.88 46.49 56.97 48.11 Nymph extract 2,000 27.05 43.01 28.3 32.79 Ginseng seed extract 2,000 56.91 44.51 52.27 51.23 Postia jaet (control) 250 40 55.86 41.15 45.67 No treatment - 25.81 37.61 32.12 31.85

Streptomyces Enhancement N-Sys of the present invention (Streptomyces To investigate the inhibitory effect of BIG11003 strain, nimdae and ginseng seed extracts on the hatching rate of root nematode eggs, roots of root-knot nematode were stained with 0.15% phloxine B staining reagent and stained oocysts were collected Respectively. The collected oocysts were washed with 1% sodium hypochlorite solution, and 200 mesh and 500 mesh sieves were used to obtain root nodule nematodes. The obtained eggs were allowed to stand at room temperature for 1 hour and then inoculated with 100 ng / ml of nematodes in a 24-well plate. The hatching rate of root - knot nematode eggs was observed for 2 weeks by inoculation with BIG11003 strain, nimdae extract and ginseng seed extract. The hatching rate of nematodes was determined by counting the number of larvae. As a result, it was confirmed that mobility decreased in all the treatments. In particular, the nimex extract and the nisseum seed extract showed egg hatching rates similar to those of the pesticide zoster, (Table 2).

Inhibitory effect of egg hatching on root hull Processing contents Treatment concentration (ppm) Egg hatching rate (%) Ⅰ repeat Ⅱ repeat Ⅲ repeat Average S. koyangensis BIG11003 strain 2,000 71.18 94.9 34.78 66.95 Nymph extract 2,000 17.37 12.76 7.43 12.52 Ginseng seed extract 2,000 8.72 14.95 11.86 11.85 Postia jaet (control) 250 15.79 7.69 12.41 11.97 No treatment - 87.78 60.48 70.06 72.44

Example  3. The eelworm  Antiseptic Root-knot nematode  Prevention and Control Effect

New Streptomyces Enhancement N-Sys of the present invention (Streptomyces The present invention relates to a method for preventing and controlling nematode root nodule in a nematode of the present invention, wherein the nematode inhibitor is selected from the group consisting of BIG11003 and plant-derived nematode extracts.

The nematocidal composition ratio of the present invention Material name Composition ratio (%) S. koyangensis BIG11003 strain 10 Nymph extract 20 Ginseng seed extract 30 water 40 Sum 100

In order to confirm the effects of the nematocide of the present invention on the nematode effect on the root nematode, the effect of decreasing the egg hatching rate was investigated. As a result, when the concentration of 1,000 ppm was treated, (Table 4).

Effect of the nematocide of the present invention on the hatching of egg root nematodes Treatment Treatment concentration (ppm) Hatching rate of nematode eggs (%) No treatment 0 72.44 Postia jaet (control) 250 11.97 The nematode controlling agent 2000 31.52 1000 19.13 500 20.98 250 21.37

In order to examine the root-knot nematode prevention effect by the pretreatment of the nematocarbons of the present invention, the nematocarbons of the present invention and the post-thiazides (nematicide liquor) were injected into pots planted with tomato seedlings at intervals of 300 ml per pot After the treatment, 1,200 nematode larvae were inoculated. The roots of tomato seedlings cultivated for 10 weeks were compared. As a result, the weight of tomato roots treated with the nematocarbons of the present invention at the concentrations of 1,000 ppm and 2,000 ppm was similar to that of the roots treated with the control zeotype root (Fig. 3) and root weight (Table 5), and the growth of the upper part (Fig. 4) was also smoothly observed.

The root weight of the tomato seedlings pretreated with the nematocide of the present invention Treatment Treatment concentration (ppm) Root weight (g) No treatment 0 5.012 Postia jaet (control) 250 11.414 The nematode controlling agent 2000 10.968 1000 10.302

In order to examine the effect of the nematode controlling agent of the present invention on the crops infected with root-knot nematode, 1200 seeds of root-knot nematode larvae were inoculated on the tomato seedlings to induce root canals and oocysts, Potashia zeit (Nematodes liquor) as a control and control was treated with 300 ml per pot, and rooting index and ovarian capsule index of tomato seedlings cultivated for 6 weeks were counted. As a result, when the nematocyst of the present invention was treated at a concentration of 1,000 ppm in the seedlings formed with the root and oocysts, the inhibitory effect was higher than that of the control seedy zygote, and the rootlets were reduced by 66% and the oocysts by 56% (FIG. 5 and Table 6), and it was confirmed that the growth of the above-ground portion (FIG. 4) was smoothly performed by the nematocyst treatment of the present invention (FIG. 6).

Effect of the Nematode Controlling Agent of the Present Invention on the Root and Oocyte Index of Crops Infected with Root-knot Nematode Treatment Treatment concentration (ppm) Root Index (%) Oanang index Non-treatment 0 100 497.3 Postia jaet (control) 250 70 216.0 The nematode controlling agent 1000 46.6 121.7

Agricultural Biotechnology Research Institute KACC91843P 20130711

Claims (7)

Streptomyces having protease-producing ability isolated from the soil ( Streptomyces BAY11003 strain (KACC 91843P). The Streptomyces enhancens BIG11003 strain according to claim 1, wherein the strain has a live nematode activity against a root nematode ( Meloidogyne sp.). delete A composition for preventing or treating infection of a root nodule ( Meloidogyne sp.) Comprising the strain of claim 1 or 2 and a pesticide acceptable carrier. A method for preventing or treating infection of a root nodule ( Meloidogyne sp.) By treating the composition of claim 4 to a plant part, soil or seed. A control agent for a root nodule ( Meloidogyne sp.) Containing the composition of claim 4 as an active ingredient. The method of claim 6, wherein the root-knot nematode control agent's (Azadirachta indica) extracts and neosam (Sophora angustifolia) root-knot nematode, characterized in that further comprises one or more of the extract (Meloidogyne sp.) The control.

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